Method for polymerizing a monomer solution within a cavity to generate a smooth polymer surface

ABSTRACT

In preferred embodiments, the present invention relates to methods for polymerizing a monomer solution within a cavity covered by a porous membrane to generate a smooth polymer surface. More specifically, the method can be used to provide a medical device or sensor with a smooth polymer surface.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/026,396, filed Feb. 5, 2008 and entitled “Method for Polymerizing aMonomer Solution within a Cavity to Generate a Smooth Polymer Surface,”which claims the benefit of U.S. Provisional Patent Application No.60/888,475, filed Feb. 6, 2007. All of the above referenced applicationsare incorporated by reference herein in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

In preferred embodiments, the present invention relates to methods forpolymerizing a monomer solution within a cavity such that the outersurface of the cavity has a smooth surface. More specifically, methodsare disclosed for making a sensor comprising functional chemistryimmobilized within a polymeric matrix disposed within a cavity along thesensor, wherein the sensor has a smooth outer surface.

2. Description of the Related Art

Polymers are widely used for coating surfaces in a wide variety ofapplications. For example, polymers are used to coat metals, fabrics,paper and glass to provide corrosion resistance, water resistance andinsulation. With respect to biomedical applications, polymers can beused to increase the biocompatibility of a surface or to provide otherdesirable properties, such as immobilizing functional chemistries forintravascular deployment.

A variety of surface coating methods exist. One method involves dippingthe surface to be treated in a solution or emulsion of a polymer andthen either letting it dry or transferring the surface into acoagulation bath which is capable of extracting the solvent from thepolymer solution. If the coat needs to be made thicker, the process canbe repeated to add another layer of polymer to the coated surface.

In another method, the polymer is formed into a powder that iselectrostatically sprayed onto a neutrally or oppositely chargedsurface. The charged polymer powder particles electrostatically adhereonto the surface. Heat treatment of the powdered surface cures andfinishes the coated surface.

In another method, the surface is heated and immersed in a fluidized bedof powdered polymer particles. The fluidized bed of polymer particles isformed by aerating a bed of polymer particles with a gas. The powderadheres to the heated surface, which is then removed from the fluidizedbed and further heated to cure and finish the coated surface.

There remains an unmet need for methods of making an analyte sensor, byimmobilizing function chemistries in a polymeric matrix within a cavityin the sensor, such that the chemistries retain their functionality andwherein the outer surface of the sensor is smooth and non-thrombogenic.

SUMMARY OF THE INVENTION

A method is disclosed for making an analyte sensor having a smooth outersurface. The method comprises the steps of: providing an optical fibercomprising a cavity covered by a membrane having pores; loading thecavity and the membrane pores with a solution comprising polymerizablemonomers, an analyte indicator system and a polymerization initiator;and initiating polymerization of the monomers.

In a preferred variation, prior to initiating polymerization, the loadedcavity and membrane pores are coated with wax and the solution isdeoxygenated. In a further variation, the wax coating is removed afterpolymerization is completed to leave a smooth outer surface, wherein theanalyte indicator system is immobilized within the cavity. Removing thewax may comprise contacting the wax with an organic solvent. Preferablythe organic solvent is hexane. In another variation, the step ofremoving the wax may further comprise application of ultrasonic energy.

In one embodiment, the indicator system comprises a fluorophore and ananalyte binding moiety.

In one embodiment, the loading step comprises vacuum filling.

In one embodiment, initiating polymerization comprises application of asecond initiator selected to undergo a redox reaction with the firstinitiator.

In other embodiments, initiating polymerization comprises application ofthermal or radiation (e.g., UV) energy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cut-away view of a sensor where a portion of the porousmembrane sheath is cut away to expose the optical fiber and hydrogelbeneath the membrane.

FIG. 2 is a cross-sectional view along a longitudinal axis of a sensorwith a hydrogel disposed distal the optical fiber.

FIG. 3A is a cross-sectional view of a portion of the porous membranesheath, optical fiber and cavity before polymerization.

FIG. 3B is a cross-sectional view of a portion of the porous membranesheath, optical fiber and cavity after polymerization.

FIG. 4 is a cut-away view of the sensor shown in FIG. 1 disposed in asecond solution.

FIG. 5 is a cut-away view of the sensor shown in FIG. 1 covered with acoat of wax.

FIG. 6A is a side view of the optical fiber showing the arrangement ofthe cavities on the optical fiber.

FIG. 6B is a side view of the optical fiber showing the arrangement ofalternatively shaped cavities on the optical fiber.

FIG. 6C is a side view of the optical fiber showing the arrangement ofyet another alternatively shaped cavities on the optical fiber.

DETAILED DESCRIPTION

FIG. 1 shows a sensor 2 comprising an optical fiber 10 with a distal end12 disposed in a porous membrane sheath 14. The optical fiber 10 hascavities 6, such as holes, in the fiber optic wall that can be formedby, for example, mechanical means such as drilling or cutting. Thecavities 6 in the optical fiber 10 can be filled with a suitablecompound, such as a polymer. In some embodiments, the polymer is ahydrogel 8. In other embodiments of the sensor 2 as shown in FIG. 2, theoptical fiber 10 does not have cavities 6, and instead, the hydrogel 8is disposed in a space distal to the distal end 12 of the optical fiber10 and proximal to the mirror 23. In some embodiments, the sensor 2 is aglucose sensor. In some embodiments, the glucose sensor is anintravascular glucose sensor.

In some embodiments, the porous membrane sheath 14 can be made from apolymeric material such as polyethylene, polycarbonate, polysulfone orpolypropylene. Other materials can also be used to make the porousmembrane sheath 14 such as zeolites, ceramics, metals, or combinationsof these materials. In some embodiments, the porous membrane sheath 14is microporous and has a mean pore size that is less than approximatelytwo nanometers. In other embodiments, the porous membrane sheath 14 ismesoporous and has a mean pore size that is between approximately twonanometers to approximately fifty nanometers. In still otherembodiments, the porous membrane sheath 14 is macroporous and has a meanpore size that is greater than approximately fifty nanometers.

In some embodiments as shown in FIG. 2, the porous membrane sheath 14 isattached to the optical fiber 10 by a connector 16. For example, theconnector 16 can be an elastic collar that holds the porous membranesheath 14 in place by exerting a compressive force on the optical fiber10. In other embodiments, the connector 16 is an adhesive or a thermalweld.

In some embodiments as shown in FIG. 1, a mirror 23 and thermistor 25can be placed within the porous membrane sheath 14 distal the distal end12 of the optical fiber 10. Thermistor leads 27 can be made to run in aspace between the optical fiber 10 and porous membrane sheath 14.Although a thermistor 25 is shown, other devices such as a thermocouple,pressure transducer, an oxygen sensor, a carbon dioxide sensor or a pHsensor for example can be used instead.

In some embodiments as shown in FIG. 2, the distal end 18 of the porousmembrane sheath 14 is open and can be sealed with, for example, anadhesive 20. In some embodiments, the adhesive 20 can comprise apolymerizable material that can fill the distal end 18 and then bepolymerized into a plug. Alternatively, in other embodiments the distalend 18 can be thermally welded by melting a portion of the polymericmaterial on the distal end 18, closing the opening and allowing themelted polymeric material to resolidify. In other embodiments as shownin FIG. 1, a polymeric plug 21 can be inserted into the distal end 18and thermally heated to weld the plug to the porous membrane sheath 14.Themoplastic polymeric materials such as polyethylene, polypropylene,polycarbonate and polysulfone are particularly suited for thermalwelding. In other embodiments, the distal end 18 of the porous membranesheath 14 can be sealed against the optical fiber 10.

After the porous membrane sheath 14 is attached to the optical fiber 10and the distal end 18 of the porous membrane sheath 14 is sealed, thesensor 2 can be vacuum filled with a first solution 15 comprising amonomer, a crosslinker and a first initiator. Vacuum filling of apolymerizable solution through a porous membrane and into a cavity in asensor is described in detail in U.S. Pat. No. 5,618,587 to Markle etal.; incorporated herein in its entirety by reference thereto. The firstsolution 15 is allowed to fill the cavity 6 within the optical fiber 10.In addition, as shown in FIG. 3A, the first solution 15 can also fillthe void volume within the porous membrane sheath 14 which comprisespores 17 and channels 19 that are capable of being filled with the firstsolution 15.

In some embodiments, the first solution 15 is aqueous and the monomer,the crosslinker and the first initiator are soluble in water. Forexample, in some embodiments, the monomer is acrylamide, the crosslinkeris bisacrylamide and the first initiator is ammonium persulfate. Inother embodiments, the monomer is dimethylacrylamide orN-hydroxymethylacrylamide. By increasing the concentrations of themonomer and/or crosslinker, the porosity of the resulting gel can bedecreased. Conversely, by decreasing the concentrations of the monomerand/or crosslinker, the porosity of the resulting gel can be increased.Other types of monomers and crosslinkers are also contemplated. In otherembodiments, the first solution 15 further comprises an analyteindicator system comprising a fluorophore and an analyte binding moietythat functions to quench the fluorescent emission of the fluorophore byan amount related to the concentration of the analyte. In someembodiments, the fluorophore and analyte binding moiety are immobilizedduring polymerization, such that the fluorophore and analyte bindingmoiety are operably coupled. In other embodiments, the fluorophore andanalyte binding moiety are covalently linked. The indicator systemchemistry may also be covalently linked to the polymeric matrix. Somepreferred fluorophores include HPTS-triLys-MA and HPTS-triCys-MA, andsome preferred analyte binding quencher moieties include 3,3′-oBBV andderivatives thereof; these and other fluorophores and quenchers aredescribed in detail in U.S. Provisional Application No. 60/833,081 andU.S. patent application Ser. No. 11/671,880, entitled OPTICALDETERMINATION OF pH AND GLUCOSE, filed on the same day as the presentapplication; these disclosures are incorporated herein by reference intheir entirety.

In some embodiments as shown in FIG. 4, after the sensor 2 is filledwith the first solution 15, the optical fiber 10 and the first solution15 filled porous membrane sheath 14 and cavity 6 are transferred to andimmersed into a second solution 24 comprising a second initiator. Insome embodiments, the second solution 24 is aqueous and the secondinitiator is tetramethylethylenediamine (TEMED). In some embodiments,the second solution 24 further comprises the same fluorescent dye and/orquencher found in the first solution 15 and in substantially the sameconcentrations. By having the fluorescent dye and quencher in both thefirst solution 15 and the second solution 24, diffusion of fluorescentdye and quencher out of the first solution 15 and into the secondsolution 24 can be reduced. In some embodiments where a second solution24 is used, the second solution 24 further comprises monomer insubstantially the same concentration as in the first solution 15. Thisreduces diffusion of monomer out of the first solution 15 by reducingthe monomer gradient between the first solution 14 and the secondsolution 24.

In some embodiments as shown in FIG. 3A, at or approximately at theinterface 26 between the first and second solutions 15 and 24, the firstinitiator and the second initiator can react together to generate aradical. In some embodiments, the first initiator and the secondinitiator react together in a redox reaction. In other embodiments, theradical can be generated by thermal decomposition, photolytic initiationor initiation by ionizing radiation. In these other embodiments, theradical may be generated anywhere in the first solution. Once theradical is generated, the radical can then initiate polymerization ofthe monomer and crosslinker in the first solution 15.

As shown in FIG. 3B, when the radical is generated via a redox reactionas described herein, the polymerization proceeds generally from theinterface 26 to the interior of the porous membrane sheath 14 andtowards the cavity 6 in the optical fiber 10. Rapid initiation ofpolymerization at the interface 26 can help reduce the amount of firstinitiator that can diffuse from the first solution 15 and into thesecond solution 24. Reducing the amount of first initiator that diffusesout of the first solution 15 helps reduce polymerization of monomeroutside the porous membrane sheath 14 which helps in forming a smoothexternal surface. Polymerization of the monomer and crosslinker resultsin a hydrogel 8 that in some embodiments substantially immobilizes theindicator system, forming the sensor 2.

In some embodiments, the first solution 15 is aqueous while the secondsolution 24 is organic. In some of these embodiments, the monomer issubstantially soluble in the aqueous first solution 15 but is notsubstantially soluble in the organic second solution 24. Because themonomer is not soluble in the second solution 24, it will not diffuseinto the second solution 24 and polymerization occurs within the firstsolution 15. Interfacial tension at the interface 26 between the firstsolution 15, the second solution 24 and the surface porous membranesheath 14 can affect the amount of penetration of the organic secondsolution 24 into the pores 17 of the porous membrane sheath 14.Penetration of the second solution 24 into the porous membrane sheath 14reduces the smoothness of the porous membrane sheath 14 afterpolymerization because the resulting hydrogel 8 is not flush with theopening of the pores 17.

In some embodiments, a surfactant that interacts with the surface of theporous membrane sheath 14 and the aqueous first solution 15 is added tothe hydrophobic second solution 24. By changing the amount and type ofsurfactant added, the interfacial tension can be changed so that theinterface 26 between the aqueous first solution 15 and the organicsecond solution 24 forms at the opening of the pores 17 in the porousmembrane sheath 14. If the interface 26 forms at the opening of thepores 17, the resulting hydrogel 8 will be flush with the opening of thepores 17, resulting in a smooth surface.

In some embodiments, the porous membrane sheath 14 can be pretreated tochange its hydrophilicity. Changing the hydrophilicity of the porousmembrane sheath 14 changes the surface tension between the porousmembrane sheath 14 and the first solution 15 and the second solution 24.For example, the porous membrane sheath 14 can be plasma etched toincrease its hydrophilicity, which in some embodiments helps inmaintaining the interface between the first solution 15 and the secondsolution at the opening of the pores 17.

In some embodiments having an organic second solution, the firstinitiator in the first solution 15 is a thermal initiator that generatesradicals upon thermal decomposition. Use of a thermal initiatorgenerally removes the need for a second initiator in the second solution24. In some embodiments, the thermal initiator decomposes and generatesradicals below a temperature of 55 degrees Celsius. Use of a thermalinitiator is particularly suitable for thermally stable dyes, quenchers,monomers and crosslinkers.

In some embodiments as shown in FIG. 5, in order to reduce water lossand to facilitate deoxygenation, after the porous membrane sheath 14 andcavity 6 are loaded with the first solution 15 comprising a thermalinitiator, the porous membrane sheath 14 is coated with a wax 28. Thiscan be accomplished by dipping the sensor 2 into liquid wax 28, which isallowed to harden around the porous membrane sheath 14.

In some embodiments, the wax 28 has a melting point above the thermalinitiation temperature of the thermal initiator. Therefore, in order toreduce the likelihood of initiation during the wax coating process, thesensor 2 can be dipped and withdrawn from the liquid wax rapidly,thereby reducing the exposure of the initiator to the elevatedtemperature of the liquid wax 28. If desired, the sensor 2 comprisingthe first solution 15 and thermal initiator can be cooled or chilled sothat the exposure to the hot wax 28 does not result in initiation and sothe wax solidifies rapidly on the sensor surface. In addition, chillingthe first solution 15 can be done during the loading of the firstsolution 15 into the sensor 2, which can be a relatively long process insome embodiments, thereby reducing the premature decomposition of theinitiator which can result in early initiation of polymerization. Also,the thickness of the wax coating can be controlled in part by thetemperature of the sensor 2 and/or the temperature of the liquid wax 28when the sensor 2 is dipped into the liquid wax 28. The colder thesensor 2, the thicker the wax coating, and the hotter the liquid wax 28,the thinner the wax coating. After the coated sensor 2 is withdrawn fromthe liquid wax bath, the wax 28 is allowed to harden. In someembodiments, hardening of the wax coating can be facilitated by dippingthe coated sensor 2 in a cold water bath. Additional coats of wax 28 canbe put on the sensor 2 by simply dipping the wax coated sensor 2 intothe liquid wax bath for an additional coat of wax.

In preferred embodiments, the coating of wax 28 is substantiallyimpermeable to water and water vapor but permeable to oxygen. Thisreduces the loss of water from the first solution 15 that can occurthough net diffusion of water or water vapor out of the first solution15 while allowing the deoxygenation of the first solution 15.

In some embodiments, deoxygenation is performed by placing the wax 28coated sensor 2 into an aqueous bath while bubbling a gas, such asnitrogen, in the bath. The oxygen in the first solution 15 diffuses intothe bath and is carried away by the nitrogen gas. The aqueous bath canbe made to have the same osmolarity and water vapor pressure as thefirst solution, thereby reducing the loss of water from the firstsolution 15 within the sensor 2. Because of the multiple measures usedto reduce water loss out of the first solution 15, the sensor 2 canremain in the deoxygenation bath for extended periods of time, such asfor example 1, 2, 4, 8, 12, 16 or 24 hours or more. In otherembodiments, deoxygenation is substantially complete in less than 24hours and therefore the next step in the polymerization process can beinitiated in less than 24 hours. Deoxygenation reduces the formation ofperoxides during the polymerization process which interfere with thepolymerization of the monomer and the performance of the dye andquencher. In addition, because oxygen can function as an inhibitor ofthe polymerization reaction, the presence of oxygen in the firstsolution 15 can reduce the efficiency of the polymerization reaction byreducing the conversion of monomer into polymer and by inhibiting theinitiation of the polymerization reaction.

In some embodiments, after the deoxygenation step, polymerization can beinitiated by heating the sensor 2 and first solution 15 comprising thethermal initiator above the thermal initiation temperature but below themelting point of the wax. For example, in some embodiments, the sensor 2is heated to 37 degrees Celsius for 24 hours. After polymerization ofthe hydrogel is complete, the wax can be removed from the sensor 2,e.g., by immersing in hexane and optionally including application ofultrasound energy. In other embodiments, a different solvent can be usedinstead of hexane. For example, other alkane hydrocarbons such aspentane and heptane and mixtures of these alkane hydrocarbons would alsobe suitable to remove the wax. In addition, it should be understood thata hexane solvent can comprise a mixture of hexane isomers, or moregenerally, an alkane solvent can comprise a mixture of the alkaneisomers. The hexane wash and ultrasound may be repeated as necessary.The sensor thereby stripped of its wax coating is optionally transferredinto an alcohol bath, and preferably finally to a water bath.

In another embodiment, the first solution 15 further comprises a secondmonomer that has at least one functional group. After polymerizationinto a hydrogel, different chemical moieties can be attached to thefunctional groups. Examples of chemical moieties with useful traits aremolecules with anti-thrombogenic properties or anti-immunogenicproperties.

In another embodiment, after the first solution 15 is loaded into theporous membrane sheath 14 and cavity 6, the optical fiber 10 and firstsolution 15 filled porous membrane sheath 14 are removed from the firstsolution 15 and polymerization is initiated in air without the use of asecond solution 24. Photolytic initiation or ultraviolet light (UV)initiation can be used in these embodiments by selecting aphotosensitizer or a UV initiator, as appropriate. Alternatively, insome embodiments, the monomer or crosslinker itself may generateradicals upon absorption of visible light or UV radiation. In someembodiments, it is desirable to have the air and first solutioninterface at the opening of the pores. In some embodiments, a surfactantis added to the first solution 15 so that the interfacial tensionbetween the first solution 15 and the surface of the porous membranesheath 14 results in the interface forming at the opening of the pores.Additionally, UV initiation may be combined with the wax coatingprocedure described above by selecting a wax that transmits UV.Additionally, UV initiation may be done with the use of a secondsolution 24. For example the second solution 24 may be chosen so as tominimize water loss during the UV polymerization process.

In some embodiments, the selection of monomer and crosslinker results ina polymer with acidic properties. For example, a monomer with an acidicfunctional group such as a phenol group can result in a polymer withacidic properties. In other embodiments, the selection of monomer andcrosslinker results in a polymer with basic properties. For example, amonomer with a basic functional group such as an amine group can resultin a polymer with basic properties. In additional embodiments, theselection of monomer and crosslinker results in a polymer withsubstantially neutral properties.

In some embodiments, polymerization is carried out at temperatures below50° C. For dyes and quenchers that are unstable at high temperatures,polymerization at low temperatures is desirable. In other embodiments,polymerization is carried out at temperatures above 50° C.Polymerization at elevated temperatures is suitable for thermally stabledyes, quenchers, and reaction components.

In some embodiments, the first solution 15 is organic and the firstinitiator, the monomer and the crosslinker are soluble in organicsolvents. In these embodiments, the second solution 24, if present, isaqueous in some embodiments and organic in other embodiments. By havingan aqueous second solution 24, diffusion of organic soluble firstinitiator, monomer and crosslinker from the first solution 15 and intothe second solution 24 is reduced. When the first solution 15 isorganic, the method of polymerization is generally more flexible. Forexample, in some embodiments, polymerization proceeds as an additionreaction. In other embodiments, polymerization proceeds as a ringopening reaction or a condensation reaction. In some embodiments,radical polymerization is used, and in other embodiments anionic orcationic polymerization is used.

In some embodiments, the monomer is not polymerized in a solvent, butinstead, undergoes bulk polymerization. In these embodiments, themonomer is generally a liquid and the initiator, crosslinker, dye and/orquencher are generally soluble in the monomer, allowing thepolymerization to be carried out without a solvent.

In some embodiments, the monomers and/or crosslinkers polymerize to forma hydrophilic polymer. In other embodiments, the monomers and/orcrosslinkers polymerize to form a hydrophobic polymer. In someembodiments, the polymer is gas permeable.

In some embodiments, the polymerization can be accomplished at a basicpH by using an appropriate initiator combination. For example in someembodiments, the first initiator is TEMED, which is basic, and thesecond initiator is ammonium persulfate. This is advantageous when thedye and quencher are more soluble at a basic pH.

In other embodiments, the polymerization can be accomplished at anacidic pH by using an appropriate initiator combination. For example insome embodiments, the first initiator is ascorbic acid, which is acidic,and the second initiator is ammonium persulfate. Another example wouldbe to use ascorbic acid with Fenton's reagent, where Fenton's reagentcomprises hydrogen peroxide and a ferrous salt. Another example of aninitiator pair is t-butyl hydroperoxide and sodium formaldehydesulfoxilate.

In some embodiments as shown in FIG. 6A, the cavities 6 arecylindrically shaped holes that are spirally arranged on the opticalfiber 10. The spiral arrangement of the cavities 6 can be accomplishedby, for example, drilling a first cavity 6 in the optical fiber 10 alonga transverse axis of the fiber 10, rotating the optical fiber 10 by aset amount between 0 and 360 degrees, and drilling the second cavity 6distal the first cavity 6. To add an additional cavity 6, the fiber 10is again rotated by the set amount and the additional cavity 6 isdrilled distal the previous cavity 6. By offsetting the cavities 6, amore complete coverage of the cross-sectional area of the optical fiber10 can be obtained, thus increasing the likelihood that excitation lightpassing through the optical fiber 10 will irradiate a sufficient andpreferably an optimal amount of the indicator system immobilized withinthe hydrogel in the cavities.

In some embodiments, the cavity 6 is drilled completely through theoptical fiber 10. In other embodiments, the cavity 6 is drilledpartially through the optical fiber 10. In embodiments where the cavity6 is drilled partially through the optical fiber 10, the cavity 6 canpenetrate approximately halfway through the fiber 10, less than halfwaythrough the fiber 10, and more than halfway through the fiber 10.

In some embodiments, the cavities 6 are not cylindrically shaped, butinstead are rectangular-shaped as shown in FIG. 6B or wedge-shaped asshown in FIG. 6C. Other cavity 6 shapes are contemplated, such ashemispherical or a spiral or helical cut that winds longitudinally downthe fiber 10, and the embodiments disclosed herein are not meant to beexhaustive. Indeed, any cavity geometries may be used in accordance withaspects of the invention.

While a number of preferred embodiments of the invention and variationsthereof have been described in detail, other modifications and methodsof using and medical applications for the same will be apparent to thoseof skill in the art. Accordingly, it should be understood that variousapplications, modifications, and substitutions may be made ofequivalents without departing from the spirit of the invention or thescope of the claims.

1. A method for making an analyte sensor having a smooth outer surface,comprising: providing an optical fiber comprising a cavity covered by amembrane having pores; loading the cavity and the membrane pores with asolution comprising polymerizable monomers, an analyte indicator systemand a polymerization initiator; and initiating polymerization of saidmonomers.
 2. The method of claim 1, wherein prior to initiatingpolymerization, the loaded cavity and membrane pores are coated with waxand the solution is deoxygenated.
 3. The method of claim 2, wherein thewax coating is removed after polymerization is completed to leave asmooth outer surface, wherein the analyte indicator system isimmobilized within the cavity.
 4. The method of claim 3, whereinremoving the wax comprises contacting the wax with an organic solvent.5. The method of claim 4, wherein the organic solvent is an alkane. 6.The method of claim 5, wherein the alkane is hexane.
 7. The methods ofclaim 3, wherein removing the wax further comprises application ofultrasonic energy.
 8. The method of claim 1, wherein the indicatorsystem comprises a fluorophore and an analyte binding moiety.
 9. Themethod of claim 1, wherein the loading step comprises vacuum filling.10. The method of claim 1, wherein initiating polymerization comprisesapplication of a second initiator selected to undergo a redox reactionwith the first initiator.
 11. The method of claim 1, wherein initiatingpolymerization comprises application of thermal or radiation energy.